This invention is concerned with a multivalent pneumococcal vaccine consisting of purified pneumococcal capsular polysaccharide with the "C" polysaccharide substantially absent. This invention is also concerned with the specific purification of each of 10 pneumococcal types which by Danish designation are types 3, 5, 9V, 10A, 11A, 15B, 17F, 19A, 22F and 33F to yield the purified immunogenic polysaccharides of the invention. Other pneumococcal types, found in the multivalent pneumococcal vaccine of this invention, which by Danish designation are types 1, 2, 4, 6A, 6B, 7F, 8, 9N, 12F, 14, 18C, 19F, 20, 23F, and 25, may be purified as shown in U.S. Pat. No. 4,242,501.
Pneumococcal cultures of each type useful in this invention are stored and available worldwide from a great number of culture libraries. The American Type Culture Collection (ATCC), 12301 Parklawn Dr., Rockville, Md., U.S.A. 20852, lists all of the pneumococcal types of this invention as being freely available.
The 1978 ATCC catalogue designates these types as follows: (See Table I)
TABLE I ______________________________________ Danish Type U.S. Catalogue Nomenclature Nomenclature Number ______________________________________ 1 1 6301 2 2 6302 3 3 6303 4 4 6304 5 5 6A 6 6306 6B 26 6326 7F 51 10351 8 8 6308 9N 9 6309 9V 68 10A 34 11A 43 12F 12 6312 14 14 6314 15B 54 17F 17 18C 56 10356 19A 57 19F 19 6319 20 20 6320 22F 22 23F 23 6323 25 25 6325 33F 70 ______________________________________
The critical step in the preparation of a vaccine is purification of the immunogenic material such that extraneous material is removed without loss of those properties of the retained material that will cause the appropriate antibody production. Such properties of polysaccharides appear to reside in the retention of what may be termed the "native state configuration" of the polysaccharides.
Among those materials to be separated from the polysaccharides are proteins, nucleic acids, and "C" polysaccharide. "C" polysaccharide is found in high concentration in Danish designation pneumococcal types 4, 7F and 14, and may be separated therefrom as shown in U.S. Pat. No. 4,242,501.
Nucleic acids (which absorb light at 260 MU) are difficult to reduce to a satisfactory level in preparations of pneumococcal polysaccharides. This problem is in contradistinction to the situation presented by meningococcal polysaccharide which is more easily purified while retaining immunogenicity. Meningococcal polysaccharide may be purified by relatively harsh methods as shown in U.S. Pat. No. 3,636,192 to Gotschlich. There are 85 specific types of pneumococcus. These types are designated by both American and Danish numbering systems. Type designations cited herein are to the Danish numbering. Each type appears to require a particular method for eliminating contaminants, but no single method is applicable to all types of pneumococcal polysaccharides. Further the specific proper method appears to be unpredictable. As exemplary of the different procedures used to purify various pneumococcal polysaccharides, some require a large quantity of ethanol for precipitation which can be partially separated from nucleic acids by fractional precipitation as the nucleic acids are precipitated in the lower alcohol ranges using 3A alcohol. 3A alcohol is 5% absolute methanol and 95% absolute ethanol. Absolute ethanol would behave in an essentially identical manner and is considered fully equivalent. Throughout this specification the term "alcohol" will designate 3A alcohol unless otherwise specified.
With other types, polysaccharides are precipitated in the 30-50% range, thus alcohol is not effective as a separatory precipitant. In contrast, other types can be separated from nucleic acids by carefully controlled amounts of protamine sulfate. With these types, at an optimal concentration of protamine sulfate (0.02-0.20%), nucleic acids are precipitated and can be pelleted by high speed centrifugation. However, any excess protamine sulfate in the system beyond the minimum amount required to precipitate the constituent nucleic acid will additionally precipitate the polysaccharide. An example of another type of purification of pneumococcal polysaccharide is presented by the purification often used for Type 3 pneumococcus, which is difficult to separate from nucleic acid. If calcium acetate is substituted for sodium acetate as the electrolyte in a solution of Type 3 pneumococcal polysaccharide, the polysaccharide can be precipitated with a minimal amount of alcohol (10-12%). However, this method sometimes allows substantial amounts of nucleic acid to remain soluble in the supernatant phase. The behavior of various pneumococcal polysaccharide types in a reaction of the polysaccharide-nucleic acid mixtures with ammonium sulfate is also variable. Some polysaccharides are precipitated by ammonium sulfate salt at 50-60% saturation whereas others are not. Type 1 polysaccharide is not precipitated with ammonium sulfate whereas Type 3 and Type 4 may be separated to some degree from nucleic acids by 50% saturation with ammonium sulfate. From the foregoing exposition and from the following references [Guy, R. C. W., How, J., Stacey, M., Heidelberger, M., J. Biol. Chem. 242: 21 (1967); Brown, R., J. Immunol. 37: 445 (1939); Glaudemans, C. P. J., Treffers, H. P., Carbohydrate Res. 4, (1967); Kabat, E. A., Exp. Immunochemistry, Charles C. Thomas, publisher, pp. 838-842 (1967)], it can be seen that there is no one satisfactory method for the removal of contaminants from pneumococcal polysaccharide applicable to all types in view of the fact that there are 85 or more types of pneumococcus and the production of a practical vaccine usually requires a multivalent vaccine comprising polysaccharide fractions from many species of pneumococcus, each retaining a relatively native state configuration.
Another contaminant of pneumococcal polysaccharide is protein. Although alcohol precipitation is effective in reducing the level of protein contamination, it is unable to reduce the contamination to a level satisfactory for a parenteral product. One method commonly employed to reduce the level of protein is to subject a mixture of pneumococcal polysaccharides and protein to organic solvents. For example, the "Sevag" procedure [Sevag, M. G. Biochem. Z., 272: 419 (1934)] involves extraction of chloroform and butanol mixtures shaken vigorously for 4-6 hours and then subjected to low speed centrifugation. Denatured protein which collects at the interface can then be separated from the aqueous phase with the polysaccharides. However, this procedure is unsatisfactory as the extraction often adversely affects the pneumococcal polysaccharides causing their breakdown, depolymerization or loss of native state configuration. The result is polysaccharides that are not effective as immunogens. Other procedures may be employed to reduce protein contamination such as ammonium sulfate precipitation and molecular sieving, but such procedures are specific to each group of proteins and peptides among the many different sizes and types of proteins in the solution. Here again the variability of the polysaccharides, depending on the strain, determines the effectiveness of the particular protein separatory step employed. Further, one may conclude that no one procedure is effective in purifying all pneumoccocal capsular polysaccharides, and prediction of the behavoir of a particular pneumococcal capsular polysaccharide appears impossible.
However, a number of methods of purifying pneumococcal capsular polysaccharides with high purity and retention of immunogenic properties have now been discovered. These purifications have been specifically directed to the purification of 10 types of pneumococcus. These types are 3, 5, 9V, 10A, 11A, 15B, 17F, 19A, 22F and 33F (Danish designation).